Compositions and methods for inhibition of cathepsins

ABSTRACT

This invention is directed to compound of Formula I and methods of using these compounds in the treatment of conditions in which modulation of a cathepsin, particularly cathepsin K or cathepsin L, will be therapeutically useful.

I. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/505,165, filed Oct. 2, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/786,142, filed Mar. 5, 2013, now U.S. Pat. No.8,877,967, which claims the benefit of U.S. Provisional PatentApplication No. 61/615,091, filed on Mar. 23, 2012, each of whichapplication is incorporated herein by reference in its entirety.

II. INTRODUCTION

A. Field

The present invention relates to compounds and methods of using thesecompounds in the treatment of conditions in which modulation of thecathepsin, particularly cathepsin K or cathepsin L, is therapeuticallyuseful.

B. Background

There are five classes of proteases including matrix metalloproteases(MMPs), cysteine proteases, serine proteases, aspartic proteases, andthreonine proteases which catalyze the hydrolysis of peptide bonds. Dueto their function in many disease states, including cancer andcardiovascular disease, proteases have become well-investigatedtherapeutic targets. Upregulation of MMPs is associated with cancermetastasis, consequently much research has been done to inhibit theiractivity. Since inhibitors of MMPs have failed to progress beyondclinical trials, interest in the other classes of proteases astherapeutic targets has grown significantly.

Cysteine protease cathepsins, members of the papain family, haverecently been validated as an important enzymatic class to target incancer research. In this family, there are eleven cathepsin enzymesknown to date in humans: B, C, F, H, K, L, O, S, V, W, and X. Cathepsinsare found in the highest concentration in cellular lysosomes, and duringcancer progression they are secreted at an increased rate and degradethe extracellular matrix and basement membrane, which aid in cancermetastasis. Cathepsins B and L have been investigated extensively, dueto their increased expression and activity in human and mouse tumors.Cathepsin K has also been the target of much research, due to its rolein bone resorption and implications in osteoporosis. Odanacatib, aninhibitor of cathepsin K developed by Merck, is currently in phase IIIclinical trials for the treatment of osteoporosis.

Cathepsin L also has a major function in intracellular lysosomalproteolysis, and in the degradation of the extracellular matrix (ECM)during the growth and metastasis of primary tumors. Despite theimportance of cathepsin L in cancer metastasis and considerable interestin the enzyme as a target for synthesis of new potential anticanceragents, there are no clinical trials testing inhibitors of cathepsin Lin cancer metastasis. This is in contrast to the application ofodanacatib to prevent bone loss in osteoporosis and cancer that hasmetastasized to bone. Odanacatib is a specific inhibitor of cathepsin K,an enzyme that is involved in degradation of the extracellular matrixproteins associated with bone resorption. Cathepsin K is a distinctenzyme in structure and function from that of cathepsin L. Smallmolecule inhibitors of cathepsin L have been previously identified,including azapenone (I), a cyanamide derivative (II), and a purinenitrile analogue (III), as well as amino acid based molecules includingan epoxide derivative (IV) and an oxocarbazate analogue (V).

Cathepsin L also has been implicated in regulatory events relating todiabetes, immunological responses, degradation of the articularcartilage matrix, and other pathological processes (Chapman et al.,1997, Annu Rev Physiol 59:63-88; Turk and Guncar, 2003 Acta CrystallogrD Biol Crystallogr 59:203-213; Maehr et al., 2005, J Clin Invest115:2934-2943; Vasiljeva et al., 2007, Curr Pharm Des 13:387-403),including osteoporosis and rheumatoid arthritis, (McGrath, 1999 Annu RevBiophys Biomol Struct 28:181-204; Turk et al., 2001 EMBO J20:4629-4633;Potts et al., 2004 Int J Exp Pathol 85:85-96; Schedel et al., 2004 GeneTher 11:1040-1047). Further, inhibition of cathepsin L has also beenshown to block Severe Acute Respiratory Syndrome (SARS) and Ebolapseudotype virus infection (Shah et al., 2010, Molecular Pharmacology78(2):319-324).

In view of the important role of cathepsins in mediating a variety ofdisease, there is an urgent need to develop potent, efficacious andpharmaceutically acceptable compounds capabe of inhibiting the activityof cathepsins L and K.

III. SUMMARY OF THE INVENTION

This invention is directed to compounds and methods of using thesecompounds in the treatment of conditions in which modulation of acathepsin, particularly cathepsin K or cathepsin L, will betherapeutically useful.

One aspect provides a compound of formula I:

wherein

-   -   X is selected from the group consisting of C(═C), CH(OR⁶) and

-   -   n is 0, 1, 2 or 3;    -   m is 0, 1, 2 or 3;    -   p is 0, 1, 2 or 3;    -   R¹ is hydrogen, C₁-C₃ alkyl, aryl or arylalkyl;    -   R² is hydrogen or C₁-C₃ alkyl;    -   each R³ and R⁵ independently is selected from the group        consisting of hydroxyl, C₁-C₃ alkyl, C₁-C₂ alkoxy, fluoro, and        chloro;    -   each R⁴ independently is selected from the group consisting of        hydroxyl, C₁-C₆ alkyl, C₁-C₆ alkoxy, halo, amino, nitro,        nitroso, and acyl; and    -   R⁶ is selected from the group consisting of hydrogen and methyl.

In certain implementations, the invention provides compounds of formulaII:

wherein X, R³, R⁴, R⁵, n, m and p are as defined above.

In other implementations, the invention provides compounds of formulaIII:

wherein X, R³, R⁴, R⁵, n, m and p are as defined above.

In one implementation, the present invention provides a compound offormula I or formula II, a solvate, or pharmaceutically acceptable saltthereof.

In another implementation, this invention provides a method ofinhibiting an activity of a cathepsin, comprising contacting thecathepsin with an amount of a compound of Formula I effective to inhibitan activity of the cathepsin.

In another implementation, this invention provides a method ofinhibiting an activity of a cathepsin, comprising contacting in vitro acathepsin K or cathepsin L with an amount of a compound of thisinvention to inhibit an activity of the cathepsin.

In another implementation, this invention provides a method ofinhibiting an activity of a cathepsin, comprising contacting in a cell acathepsin with an amount of a compound effective to inhibit an activityof the cathepsin wherein the compound is selected from the compounds ofthis invention, as described above.

In another implementation, this invention provides a method ofinhibiting a neoplasm, comprising administering to a patient sufferingfrom such neoplasm an amount of a compound of this invention effectiveto treat the neoplasm. In other implementations, the compounds describedherein can be used to treat non-neoplastic conditions such asosteoporosis, viral infections and parasites, particularly protozoalparasites. In one aspect, the present invention provides a method forinhibiting a cysteine protease involved in the infectious life cycle ofa protozoan parasite, the method comprising the step of administering tothe subject a compound described herein, said compound administered tothe subject in an amount sufficient to disrupt the infectious life cycleof a protozoan parasite. Exemplary protozoan cysteine proteases includethose required in the infectious life cycle of a trypanosome, such ascruzain or cruzipain from T. cruzi, rhodesain or brucipain from T.brucei rhodesiense, and congopain from T. congolense; a plasmodium, suchas falcipain from P. falciparum; or a leishmania, such as CPB2.8 DeltaCTE from L. mexicana.

In another implementation, this invention provides a pharmaceuticalformulation comprising a thiosemicarbazone as described above.

In another implementation, this invention provides a kit comprising athiosemicarbazone as described above, packaging, and instructions foruse.

It will be appreciated by one of skill in the art that theimplementations summarized above may be used together in any suitablecombination to generate implementations not expressly recited above andthat such implementations are considered to be part of the presentinvention.

IV. DETAILED DESCRIPTION

The invention encompasses compounds having formula I and thecompositions and methods using these compounds in the treatment ofconditions in which modulation of a cathepsin, particularly cathepsin Kor cathepsin L, is therapeutically useful.

As used herein, the following definitions shall apply unless otherwiseindicated.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbon groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.This term includes, by way of example, linear and branched hydrocarbongroups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—),isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—),sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. Acyl includes the“acetyl” group CH₃C(O)—.

“Amino” refers to the group —NH₂.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like), provided that the pointof attachment is through an atom of the aromatic aryl group. Preferredaryl groups include phenyl and naphthyl.

“Alkenyl” refers to straight chain or branched hydrocarbon groups havingfrom 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and havingat least 1 and preferably from 1 to 2 sites of double bond unsaturation.Such groups are exemplified, for example, bi-vinyl, allyl, andbut-3-en-1-yl. Included within this term are the cis and trans isomersor mixtures of these isomers.

“Alkynyl” refers to straight or branched monovalent hydrocarbon groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andSpiro ring systems. Examples of suitable cycloalkyl groups include, forinstance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyland the like.

“Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo and ispreferably fluoro or chloro.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen, and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl, imidazolyl or furyl) or multiple condensedrings (e.g., indolizinyl, quinolinyl, benzimidazolyl or benzothienyl),wherein the condensed rings may or may not be aromatic and/or contain aheteroatom, provided that the point of attachment is through an atom ofthe aromatic heteroaryl group. In one implementation, the nitrogenand/or sulfur ring atom(s) of the heteroaryl group are optionallyoxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonylmoieties. Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl,thiophenyl, and furanyl.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 15 ring atoms, including 1 to 4 heteroatoms. These ring atoms are selected from the group consisting ofnitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or moreof the rings can be cycloalkyl, aryl, or heteroaryl, provided that thepoint of attachment is through the non-aromatic ring. In oneimplementation, the nitrogen and/or sulfur atom(s) of the heterocyclicgroup are optionally oxidized to provide for the N-oxide, —S(O)—, or—SO₂— moieties.

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, dihydroindole, indazole,purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

“Nitro” refers to the group —NO₂.

“Nitroso” refers to the group —NO.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

The term “substituted,” when used to modify a specified group orradical, means that one or more hydrogen atoms of the specified group orradical are each, independently of one another, replaced with the sameor different substituent groups as defined below.

Substituent groups for substituting for one or more hydrogens (any twohydrogens on a single carbon can be replaced with ═O, ═NR⁷⁰, ═N—OR⁷⁰,═N₂ or ═S) on saturated carbon atoms in the specified group or radicalare, unless otherwise specified, —R⁶⁰, halo, ═O, —OR⁷⁰, —SR⁷⁰, —NR⁸⁰R⁸⁰,trihalomethyl, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —SO₂R⁷⁰, —SO²O⁻M⁺,—SO₂OR⁷⁰, —OSO₂R⁷⁰, —OSO₂O⁻M⁺, OSO₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂,—P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰,—C(O)O⁻M⁺, —C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰,—OC(O)R⁷⁰, —OC(S)R⁷⁰, —OC(O)O⁻M⁺, —OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰,—NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰,—NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰ is selected from thegroup consisting of optionally substituted alkyl, cycloalkyl,heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl,heteroaryl and heteroarylalkyl, each R⁷⁰ is independently hydrogen orR⁶⁰; each R⁸⁰ is independently R⁷⁰ or alternatively, two R^(80′)s, takentogether with the nitrogen atom to which they are bonded, form a 5-, 6-or 7-membered heterocycloalkyl which may optionally include from 1 to 4of the same or different additional heteroatoms selected from the groupconsisting of O, N and S, of which N may have —H or C₁-C₃ alkylsubstitution; and each M⁺ is a counter ion with a net single positivecharge. Each M⁺ may independently be, for example, an alkali ion, suchas K⁺, Na⁺, Li⁺; an ammonium ion, such as ⁺N(R⁶⁰)₄; or an alkaline earthion, such as [Ca²⁺]_(0.5), [Mg²⁺]_(0.5), or [Ba²⁺]_(0.5) (“subscript 0.5means e.g. that one of the counter ions for such divalent alkali earthions can be an ionized form of a compound of the invention and the othera typical counter ion such as chloride, or two ionized compounds of theinvention can serve as counter ions for such divalent alkali earth ions,or a doubly ionized compound of the invention can serve as the counterion for such divalent alkali earth ions). As specific examples, —NR⁸⁰R⁸⁰is meant to include —NH₂, —NH-alkyl, N-pyrrolidinyl, N-piperazinyl,4N-methyl-piperazin-1-yl and N-morpholinyl.

In a preferred implementation, a group that is substituted has 1, 2, 3,or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1substituent.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups are limited to -substituted aryl-(substitutedaryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

“Stereoisomer” and “stereoisomers” refer to compounds that have sameatomic connectivity but different atomic arrangement in space.Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers,and diastereomers.

“Tautomer” refers to alternate forms of a molecule that differ only inelectronic bonding of atoms and/or in the position of a proton, such asenol-keto and imine-enamine tautomers, or the tautomeric forms ofheteroaryl groups containing a —N═C(H)—NH— ring atom arrangement, suchas pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Aperson of ordinary skill in the art would recognize that othertautomeric ring atom arrangements are possible.

“Patient” refers to human and non-human animals, especially mammals.

“Pharmaceutically acceptable salt” refers to pharmaceutically acceptablesalts of a compound, which salts are derived from a variety of organicand inorganic counter ions well known in the art and include, by way ofexample only, sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium, and the like; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate,oxalate, and the like.

“Pharmaceutically effective amount” and “therapeutically effectiveamount” refer to an amount of a compound sufficient to treat a specifieddisorder or disease or one or more of its symptoms and/or to prevent theoccurrence of the disease or disorder. In reference to tumorigenicproliferative disorders and neoplasms, a pharmaceutically ortherapeutically effective amount comprises an amount sufficient to,among other things, cause the tumor to shrink or decrease the growthrate of the tumor.

“Solvate” refers to a complex formed by combination of solvent moleculeswith molecules or ions of the solute. The solvent can be an organiccompound, an inorganic compound, or a mixture of both. Some examples ofsolvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.

Similarly, it is understood that the above definitions are not intendedto include impermissible substitution patterns (e.g., methyl substitutedwith 5 fluoro groups). Such impermissible substitution patterns areeasily recognized by a person having ordinary skill in the art.

This invention provides novel thiosemicarbazone compounds and methods ofmaking the compound and methods of using these compounds in thetreatment of conditions in which inhibition of a cathepsin, particularlycathepsin K and/or cathepsin L, is therapeutically useful. Theseconditions include, but are not limited to, neoplasms, osteoporosis,protozoal parasite infection and viral infections. Given the severity ofand suffering caused by these conditions, it is vital that newtreatments are developed to treat these conditions.

In one implementation, the present invention provides a compound offormula I, solvates, or pharmaceutically acceptable salts thereof.

wherein

X is selected from the group consisting of C(═O), CH(OR⁶) and

-   -   n is 0, 1, 2 or 3;    -   m is 0, 1, 2 or 3;    -   p is 0, 1, 2 or 3;    -   R¹ is hydrogen, C₁-C₃ alkyl, aryl or arylalkyl;    -   R² is hydrogen or C₁-C₃ alkyl;    -   each R³ and R⁵ independently is selected from the group        consisting of hydroxyl, C₁-C₃ alkyl, C₁-C₂ alkoxy, fluoro, and        chloro;    -   each R⁴ independently is selected from the group consisting of        hydroxyl, C₁-C₆ alkyl, C₁-C₆ alkoxy, halo, amino, nitro, nitroso        and acyl; and    -   R⁶ is selected from the group consisting of hydrogen and methyl.

Preferably, n, m and p independently are zero or one. In someimplementations, R¹ and R² independently are hydrogen or methyl. Incertain implementations, both R¹ and R² are hydrogen. In otherimplementations, n is one and R³ is selected from the group consistingof hydroxyl, methyl, methoxy, and fluoro. In alternativeimplementations, p is one and R⁵ is selected from the group consistingof hydroxyl, methyl, methoxy and fluoro. In yet other implementations, mis one and R⁴ is selected from the group consisting of halo and acyl.

In other implementations, X is C(═C) or CH(OR⁶). In a preferredimplementation, X is C(═C). In certain implementations, m is zero. Inother implementations, n is zero and p is zero. In certainimplementation m is 1, and optionally R⁴ is halo or acyl. In certainpreferred implementations, R⁴ is substituted aryl-C(O)— or unsubstitutedaryl-C(O)—, more preferably R⁴ is benzoyl. In certain implementations,each of n and p, independently, is zero or one; and each of R³ and R⁵,independently, is selected from the group consisting of hydroxyl,methyl, methoxy and fluoro.

In certain implementations, the invention provides compounds of formulaII:

wherein X, R³, R⁴, R⁵, n, m and p are as defined above. Preferably, n, mand p independently are zero or one. In some implementations, both R¹and R² are hydrogen. In other implementations, n is one and R³ isselected from the group consisting of hydroxyl, methyl, methoxy, andfluoro. In alternative implementations, p is one and R⁵ is selected fromthe group consisting of hydroxyl, methyl, methoxy and fluoro. In yetother implementations, R⁴ is selected from the group consisting of haloand acyl.

In a preferred implementation, the invention provides compounds offormula II-a

wherein m is zero or one and R³, R⁴, R⁵, n, and p are as defined above.Preferably, n and p independently are zero or one. In otherimplementations, n is one and R³ is selected from the group consistingof hydroxyl, methyl, methoxy, and fluoro. In alternativeimplementations, p is one and R⁵ is selected from the group consistingof hydroxyl, methyl, methoxy and fluoro. In a preferred implementation,m is zero. In other implementations, m is one and R⁴ is selected fromthe group consisting of halo and acyl.

In yet another implementation, the invention provides compounds offormula II-b

wherein m is zero or one and R³, R⁴, R⁵, n, and p are as defined above.Preferably, n and p independently are zero or one. In a preferredimplementation, m is zero.

In other implementations, the invention provides compounds of formulaIII:

wherein X, R³, R⁴, R⁵, n, m and p are as defined above. Preferably, n, mand p independently are zero or one. In some implementations, both R¹and R² are hydrogen. In other implementations, n is one and R³ isselected from the group consisting of hydroxyl, methyl, methoxy, andfluoro. In alternative implementations, p is one and R⁵ is selected fromthe group consisting of hydroxyl, methyl, methoxy and fluoro. In yetother implementations, R⁴ is selected from the group consisting of haloand acyl.

In a preferred implementation, the invention provides compounds offormula III-a

wherein R³, R⁴, R⁵, n, m and p are as defined above. Preferably, n, mand p independently are zero or one. In other implementations, n is oneand R³ is selected from the group consisting of hydroxyl, methyl,methoxy, and fluoro. In alternative implementations, p is one and R⁵ isselected from the group consisting of hydroxyl, methyl, methoxy andfluoro. In a preferred implementation, m is zero. In otherimplementations, m is one and R⁴ is selected from the group consistingof halo and acyl.

Another aspect of the invention provides a compound selected from Table1 or Table 2, or a solvate, tautomer, stereoisomer and/orpharmaceutically acceptable salt thereof.

TABLE 1

Compound X R¹ R² (R³)_(n) (R⁴)_(m) (R⁵)_(p)  3 —C(═O) H H n = 0 m = 0 p= 0  6 —CH(OH)— H H n = 0 m = 0 p = 0  9 —C(═O) H H 3-Me m = 0 3-Me 11—C(═O) H H 2-F 5-Br 2-F 13 —C(═O) H H 4-F m = 0 4-F 15 —C(═O) H H 4-OCH₃m = 0 4-OCH₃ 17 —C(═O) H H 4-OH m = 0 4-OCH₃ 18 —C(═O) H H 4-OCH₃ m = 04-OH 19

H H 4-OH m = 0 4-OCH₃ 22 —C(═O) H H 4-OCH(CH₃)₂ m = 0 4-OCH(CH₃)₂ 23

H H 4-OCH(CH₃)₂ m = 0 4-OCH(CH₃)₂ 24

H H 4-Br m = 0 4-Br 25 —C(═O) H H n = 0 5-benzoyl p = 0 26 —C(═O) H H4-Br m = 0 4-Br 27 —C(═O) CH₃ H n = 0 m = 0 p = 0 28 —C(═O) H CH₃ n = 0m = 0 p = 0 32

H H 4-F m = 0 4-F 33

H H 4-OCH₃ m = 0 4-OCH₃ 34 —C(═O) H H 4-OH m = 0 4-OH

TABLE 2

Compound X (R³)_(n) (R⁴)_(m) (R⁵)_(p)  4 —C(═O) n = 0 m = 0 p = 0 29—C(═O) 4-F m = 0 4-F 30 —C(═O) 4-Br m = 0 4-Br 31 —C(═O) 4-OCH₃ m = 04-OCH₃

Depending upon the nature of the various substituents, thethiosemicarbazone compounds of the invention can be in the form ofsalts. Such salts include salts suitable for pharmaceutical uses(“pharmaceutically-acceptable salts”), salts suitable for veterinaryuses, etc. Such salts can be derived from acids or bases, as iswell-known in the art.

In one implementation, the salt is a pharmaceutically acceptable salt.Generally, pharmaceutically acceptable salts are those salts that retainsubstantially one or more of the desired pharmacological activities ofthe parent compound and which are suitable for administration to humans.Pharmaceutically acceptable salts include acid addition salts formedwith inorganic acids or organic acids. Inorganic acids suitable forforming pharmaceutically acceptable acid addition salts include, by wayof example and not limitation, hydrohalide acids (e.g., hydrochloricacid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitricacid, phosphoric acid, and the like. Organic acids suitable for formingpharmaceutically acceptable acid addition salts include, by way ofexample and not limitation, acetic acid, trifluoroacetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalicacid, pyruvic acid, lactic acid, malonic acid, succinic acid, malicacid, maleic acid, fumaric acid, tartaric acid, citric acid, palmiticacid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid,mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid,ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonicacid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid, 4chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, etc.),4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like.

Pharmaceutically acceptable salts also include salts formed when anacidic proton present in the parent compound is either replaced by ametal ion (e.g., an alkali metal ion, an alkaline earth metal ion or analuminum ion) or coordinates with an organic base (e.g., ethanolamine,diethanolamine, triethanolamine, N-methylglucamine, morpholine,piperidine, dimethylamine, diethylamine, triethylamine, ammonia, etc.).

The thiosemicarbazone compounds and salts thereof may also be in theform of hydrates, solvates and N-oxides, as are well-known in the art.

In another implementation, this invention provides a compound found inTable 1 or Table 2, or stereoisomer, tautomer, solvate, orpharmaceutically acceptable salt thereof.

Cysteine proteases are known to bind to their protein substrates throughantiparallel beta sheet structures. The discovery of the improved andpotent cysteine protease inhibitors of the invention has resulted fromthe judicious extension of the thiosemicarbazone functionality intorigid carbon skeletons which mimic the beta sheet conformation of thesubstrates of these proteases and conform well to their relatively rigidactive sites (McGrath, et al. J. Mol. Biol. (1995), 247: 251-259;Gillmor, et al., Protein Science, (1997), 6: 1603-1611; Choe, et al,Bioorg. Med. Chern. Lett. (2005), 13:2141-2156; and Huang et al.,Bioorg. Med. Chern. (2003), 11: 21-29). Indeed, the compounds of theinvention are surprisingly potent inhibitors of cysteine proteaseinhibition, with IC50 values at the low nanomolar level (e.g., 10 nM orless). Accordingly, the compounds of the invention may be employed inthe treatment of parasitic disease states such as malaria, leishmaniasisand trypanosomiasis (e.g., Chagas'disease) as inhibitors of parasticcysteine proteases, including the cathepsin-L like cysteine proteases(e.g., cruzain). Moreover, the compounds of the invention also find usein the treatment of other mammalian disorders (e.g., cancer andinflammatory disorders) as inhibitors of related mammalian cysteineproteases, including cathepsin L, cathepsin B, cathepsin H, cathepsin Kand cathepsin S.

The compounds described herein are potent and selective inhibitors ofcathepsin. As a consequence of this activity, the compounds can be usedin a variety of in vitro, in vivo and ex vivo contexts to inhibitcathepsin activity.

In one implementation, the method further comprises contacting thecathepsin with the compound in a cell. In another implementation, saidcontacting occurs in vivo. In another implementation, said contactingoccurs in vitro.

In another implementation, the present invention provides a method oftreating a disorder mediated by a cathepsin, comprising administering toa patient in need thereof a therapeutically effective amount of acompound effective to treat the disorder wherein the compound is acompound of formula I.

In yet another implementation, the disorder mediated by a cathepsin is acancer where a cathepsin such as cathepsin K or cathepsin L isupregulated, such as cancers of both epithelial and mesenchymal originincluding breast, brain, lung, gastrointestinal, pancreatic, colorectal,melanoma, and head and neck cancers among others. The present compoundsalso may have a therapeutic effect in tumors such as T cell leukemia,thymoma, T and B cell lymphoma (such as diffuse large B cell lymphoma ortransformed (CD20+) indolent lymphoma), colon carcinoma, prostatecancer, ovarian cancer (e.g. ovarian epithelial or primary peritonealcarcinoma) and lung carcinoma (e.g., non-small cell lung cancer orsmall-cell lung cancer).

Pharmaceutical compositions comprising the thiosemicarbazone compoundsdescribed herein can be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilization processes. The compositionscan be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically.

The thiosemicarbazone compound can be formulated in the pharmaceuticalcompositions per se, or in the form of a hydrate, solvate, N-oxide orpharmaceutically acceptable salt, as described herein. Typically, suchsalts are more soluble in aqueous solutions than the corresponding freeacids and bases, but salts having lower solubility than thecorresponding free acids and bases may also be formed.

In one implementation, this invention provides a pharmaceuticalformulation comprising a compound selected from the compounds of thisinvention, as described above.

The compounds can be provided in a variety of formulations and dosages.The compounds can be provided in a pharmaceutically acceptable formincluding, where the compound can be formulated in the pharmaceuticalcompositions per se, or in the form of a hydrate, solvate, N-oxide orpharmaceutically acceptable salt, as described herein. Typically, suchsalts are more soluble in aqueous solutions than the corresponding freeacids and bases, but salts having lower solubility than thecorresponding free acids and bases may also be formed.

In one implementation, the compounds are provided as non-toxicpharmaceutically acceptable salts, as noted previously. Suitablepharmaceutically acceptable salts of the compounds of this inventioninclude acid addition salts such as those formed with hydrochloric acid,fumaric acid, p-toluenesulphonic acid, maleic acid, succinic acid,acetic acid, citric acid, tartaric acid, carbonic acid or phosphoricacid. Salts of amine groups may also comprise quaternary ammonium saltsin which the amino nitrogen atom carries a suitable organic group suchas an alkyl, alkenyl, alkynyl or aralkyl moiety. Furthermore, where thecompounds of the invention carry an acidic moiety, suitablepharmaceutically acceptable salts thereof may include metal salts suchas alkali metal salts, e.g. sodium or potassium salts; and alkalineearth metal salts, e.g. calcium or magnesium salts.

The pharmaceutically acceptable salts of the present invention can beformed by conventional means, such as by reacting the free base form ofthe product with one or more equivalents of the appropriate acid in asolvent or medium in which the salt is insoluble, or in a solvent suchas water which is removed in vacuo or by freeze drying or by exchangingthe anions of an existing salt for another anion on a suitable ionexchange resin.

The present invention includes within its scope solvates of thethiosemicarbazone compounds and salts thereof, for example, hydrates.

The thiosemicarbazone compounds may have one or more asymmetric centers,and may accordingly exist both as enantiomers and as diastereomers. Itis to be understood that all such isomers and mixtures thereof areencompassed within the scope of the present invention.

The thiosemicarbazone compounds can be administered by oral, parenteral(e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternalinjection or infusion, subcutaneous injection, or implant), byinhalation spray, nasal, vaginal, rectal, sublingual, urethral (e.g.,urethral suppository) or topical routes of administration (e.g., gel,ointment, cream, aerosol, etc.) and can be formulated, alone ortogether, in suitable dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, excipientsand vehicles appropriate for each route of administration. In additionto the treatment of warm-blooded animals such as mice, rats, horses,cattle, sheep, dogs, cats, monkeys, etc., the compounds of the inventioncan be effective in humans.

The pharmaceutical compositions for the administration of thethiosemicarbazone compounds may conveniently be presented in dosage unitform and can be prepared by any of the methods well known in the art ofpharmacy. The pharmaceutical compositions can be, for example, preparedby uniformly and intimately bringing the active ingredient intoassociation with a liquid carrier or a finely divided solid carrier orboth, and then, if necessary, shaping the product into the desiredformulation. In the pharmaceutical composition the active objectcompound is included in an amount sufficient to produce the desiredtherapeutic effect. For example, pharmaceutical compositions of theinvention may take a form suitable for virtually any mode ofadministration, including, for example, topical, ocular, oral, buccal,systemic, nasal, injection, transdermal, rectal, vaginal, etc., or aform suitable for administration by inhalation or insufflation.

For topical administration, the compound(s) of this invention can beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art.

Systemic formulations include those designed for administration byinjection, e.g., subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection canbe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives.

Alternatively, the injectable formulation can be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, etc., before use.To this end, the active compound(s) can be dried by any art-knowntechnique, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the pharmaceutical compositions may take theform of, for example, lozenges, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets can be coated by methods well known in theart with, for example, sugars, films or enteric coatings. Additionally,the pharmaceutical compositions containing the 2,4-substitutedpyrmidinediamine as active ingredient in a form suitable for oral use,may also include, for example, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, or syrups or elixirs. Compositions intended for oral use canbe prepared according to any method known to the art for the manufactureof pharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. Tabletscontain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients can be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents(e.g., corn starch, or alginic acid); binding agents (e.g. starch,gelatin or acacia); and lubricating agents (e.g. magnesium stearate,stearic acid or talc). The tablets can be uncoated or they can be coatedby known techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed. They may also becoated by the techniques described in the U.S. Pat. Nos. 4,256,108;4,166,452; and 4,265,874 to form osmotic therapeutic tablets for controlrelease. The pharmaceutical compositions of the invention may also be inthe form of oil-in-water emulsions.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups or suspensions, or they can bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, Cremophore™ or fractionated vegetable oils); and preservatives(e.g., methyl or propyl p hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, preservatives, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound, as is well known.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For rectal and vaginal routes of administration, the active compound(s)can be formulated as solutions (for retention enemas) suppositories orointments containing conventional suppository bases such as cocoa butteror other glycerides.

For nasal administration or administration by inhalation orinsufflation, the active compound(s) can be conveniently delivered inthe form of an aerosol spray from pressurized packs or a nebulizer withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbondioxide or other suitable gas. In the case of a pressurized aerosol, thedosage unit can be determined by providing a valve to deliver a meteredamount. Capsules and cartridges for use in an inhaler or insufflator(for example capsules and cartridges comprised of gelatin) can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The pharmaceutical compositions can be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension can beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent. Among the acceptable vehicles and solvents that can be employedare water, Ringer's solution and isotonic sodium chloride solution. Thethiosemicarbazone compounds may also be administered in the form ofsuppositories for rectal or urethral administration of the drug. Inparticular implementations, the compounds can be formulated as urethralsuppositories, for example, for use in the treatment of fertilityconditions, particularly in males, e.g., for the treatment of testiculardysfunction.

According to the invention, thiosemicarbazone compounds can be used formanufacturing a composition or medicament, including medicamentssuitable for rectal or urethral administration. The invention alsorelates to methods for manufacturing compositions includingthiosemicarbazone compounds in a form that is suitable for urethral orrectal administration, including suppositories.

For topical use, creams, ointments, jellies, gels, solutions orsuspensions, etc., containing the thiosemicarbazone compounds can beemployed. In certain implementations, the thiosemicarbazone compoundscan be formulated for topical administration with polyethylene glycol(PEG). These formulations may optionally comprise additionalpharmaceutically acceptable ingredients such as diluents, stabilizersand/or adjuvants. In particular implementations, the topicalformulations are formulated for the treatment of allergic conditionsand/or skin conditions including psoriasis, contact dermatitis andatopic dermatitis, among others described herein.

According to the invention, thiosemicarbazone compounds can be used formanufacturing a composition or medicament, including medicamentssuitable for topical administration. The invention also relates tomethods for manufacturing compositions including thiosemicarbazonecompounds in a form that is suitable for topical administration.

According to the present invention, thiosemicarbazone compounds can alsobe delivered by any of a variety of inhalation devices and methods knownin the art, including, for example: U.S. Pat. No. 6,241,969; U.S. Pat.No. 6,060,069; U.S. Pat. No. 6,238,647; U.S. Pat. No. 6,335,316; U.S.Pat. No. 5,364,838; U.S. Pat. No. 5,672,581; WO96/32149; WO95/24183;U.S. Pat. No. 5,654,007; U.S. Pat. No. 5,404,871; U.S. Pat. No.5,672,581; U.S. Pat. No. 5,743,250; U.S. Pat. No. 5,419,315; U.S. Pat.No. 5,558,085; WO98/33480; U.S. Pat. No. 5,364,833; U.S. Pat. No.5,320,094; U.S. Pat. No. 5,780,014; U.S. Pat. No. 5,658,878; 5,518,998;5,506,203; U.S. Pat. No. 5,661,130; U.S. Pat. No. 5,655,523; U.S. Pat.No. 5,645,051; U.S. Pat. No. 5,622,166; U.S. Pat. No. 5,577,497; U.S.Pat. No. 5,492,112; U.S. Pat. No. 5,327,883; U.S. Pat. No. 5,277,195;U.S. Pat. Pub. No. 20010041190; U.S. Pat. Pub. No. 20020006901; and U.S.Pat. Pub. No. 20020034477.

Included among the devices which can be used to administer particularexamples of the thiosemicarbazone compounds are those well-known in theart, such as, metered dose inhalers, liquid nebulizers, dry powderinhalers, sprayers, thermal vaporizers, and the like. Other suitabletechnology for administration of particular thiosemicarbazone compoundsincludes electrohydrodynamic aerosolizers.

In addition, the inhalation device is preferably practical, in the senseof being easy to use, small enough to carry conveniently, capable ofproviding multiple doses, and durable. Some specific examples ofcommercially available inhalation devices are Turbohaler (Astra,Wilmington, Del.), Rotahaler (Glaxo, Research Triangle Park, N.C.),Diskus (Glaxo, Research Triangle Park, N.C.), the Ultravent nebulizer(Mallinckrodt), the Acorn II nebulizer (Marquest Medical Products,Totowa, N.J.) the Ventolin metered dose inhaler (Glaxo, ResearchTriangle Park, N.C.), or the like. In one implementation,thiosemicarbazone compounds can be delivered by a dry powder inhaler ora sprayer.

As those skilled in the art will recognize, the formulation ofthiosemicarbazone compounds, the quantity of the formulation delivered,and the duration of administration of a single dose depend on the typeof inhalation device employed as well as other factors. For some aerosoldelivery systems, such as nebulizers, the frequency of administrationand length of time for which the system is activated will depend mainlyon the concentration of thiosemicarbazone compounds in the aerosol. Forexample, shorter periods of administration can be used at higherconcentrations of thiosemicarbazone compounds in the nebulizer solution.Devices such as metered dose inhalers can produce higher aerosolconcentrations, and can be operated for shorter periods to deliver thedesired amount of thiosemicarbazone compounds in some implementations.Devices such as dry powder inhalers deliver active agent until a givencharge of agent is expelled from the device. In this type of inhaler,the amount of 2 thiosemicarbazone compounds in a given quantity of thepowder determines the dose delivered in a single administration. Theformulation of thiosemicarbazone is selected to yield the desiredparticle size in the chosen inhalation device.

Formulations of thiosemicarbazone compounds for administration from adry powder inhaler may typically include a finely divided dry powdercontaining thiosemicarbazone compounds, but the powder can also includea bulking agent, buffer, carrier, excipient, another additive, or thelike. Additives can be included in a dry powder formulation ofthiosemicarbazone compounds, for example, to dilute the powder asrequired for delivery from the particular powder inhaler, to facilitateprocessing of the formulation, to provide advantageous powder propertiesto the formulation, to facilitate dispersion of the powder from theinhalation device, to stabilize to the formulation (e.g., antioxidantsor buffers), to provide taste to the formulation, or the like. Typicaladditives include mono-, di-, and polysaccharides; sugar alcohols andother polyols, such as, for example, lactose, glucose, raffinose,melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, orcombinations thereof; surfactants, such as sorbitols, diphosphatidylcholine, or lecithin; or the like.

The present invention also relates to a pharmaceutical compositionincluding thiosemicarbazone compounds suitable for administration byinhalation. According to the invention, thiosemicarbazone compounds canbe used for manufacturing a composition or medicament, includingmedicaments suitable for administration by inhalation. The inventionalso relates to methods for manufacturing compositions includingthiosemicarbazone compounds in a form that is suitable foradministration, including administration by inhalation. For example, adry powder formulation can be manufactured in several ways, usingconventional techniques, such as described in any of the publicationsmentioned above and incorporated expressly herein by reference, and forexample, Baker, et al., U.S. Pat. No. 5,700,904, the entire disclosureof which is incorporated expressly herein by reference. Particles in thesize range appropriate for maximal deposition in the lower respiratorytract can be made by micronizing, milling, or the like. And a liquidformulation can be manufactured by dissolving the thiosemicarbazonecompounds in a suitable solvent, such as water, at an appropriate pH,including buffers or other excipients.

Pharmaceutical compositions comprising the thiosemicarbazone compoundsdescribed herein can be manufactured by means of conventional mixing,dissolving, granulating, dragee-making levigating, emulsifying,encapsulating, entrapping or lyophilization processes. The compositionscan be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically.

For ocular administration, the thiosemicarbazone compound(s) can beformulated as a solution, emulsion, suspension, etc. suitable foradministration to the eye. A variety of vehicles suitable foradministering compounds to the eye are known in the art. Specificnon-limiting examples are described in U.S. Pat. No. 6,261,547; U.S.Pat. No. 6,197,934; U.S. Pat. No. 6,056,950; U.S. Pat. No. 5,800,807;U.S. Pat. No. 5,776,445; U.S. Pat. No. 5,698,219; U.S. Pat. No.5,521,222; U.S. Pat. No. 5,403,841; U.S. Pat. No. 5,077,033; U.S. Pat.No. 4,882,150; and U.S. Pat. No. 4,738,851.

For prolonged delivery, the thiosemicarbazone compound(s) can beformulated as a depot preparation for administration by implantation orintramuscular injection. The active ingredient can be formulated withsuitable polymeric or hydrophobic materials (e.g., as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, e.g., as a sparingly soluble salt. Alternatively,transdermal delivery systems manufactured as an adhesive disc or patchwhich slowly releases the active compound(s) for percutaneous absorptioncan be used. To this end, permeation enhancers can be used to facilitatetransdermal penetration of the active compound(s). Suitable transdermalpatches are described in for example, U.S. Pat. No. 5,407,713.; U.S.Pat. No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168;U.S. Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No.5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S. Pat.No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475.

Alternatively, other pharmaceutical delivery systems can be employed.Liposomes and emulsions are well-known examples of delivery vehiclesthat can be used to deliver active compound(s). Certain organic solventssuch as dimethylsulfoxide (DMSO) may also be employed, although usuallyat the cost of greater toxicity.

The pharmaceutical compositions may, if desired, be presented in a packor dispenser device which may contain one or more unit dosage formscontaining the active compound(s). The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice can be accompanied by instructions for administration.

The amount of compound administered will depend upon a variety offactors, including, for example, the particular condition being treated,the mode of administration, the severity of the condition being treatedand the age and weight of the patient, the bioavailability of theparticular active compound, etc. Determination of an effective dosage iswell within the capabilities of those skilled in the art.

As known by those of skill in the art, the preferred dosage ofthiosemicarbazone compounds will also depend on the age, weight, generalhealth and severity of the condition of the individual being treated.Dosage may also need to be tailored to the sex of the individual and/orwhere administered by inhalation, the lung capacity of the individual.Dosage may also be tailored to individuals suffering from more than onecondition or those individuals who have additional conditions whichaffect lung capacity and the ability to breathe normally, for example,emphysema, bronchitis, pneumonia, respiratory infections, etc. Dosage,and frequency of administration of the compounds will also depend onwhether the compounds are formulated for treatment of acute episodes ofa condition or for the prophylactic treatment of a disorder. Forexample, acute episodes of allergic conditions, includingallergy-related asthma, transplant rejection, etc. A skilledpractitioner will be able to determine the optimal dose for a particularindividual.

For prophylactic administration, the compound can be administered to apatient at risk of developing one of the previously describedconditions. For example, if it is unknown whether a patient is allergicto a particular drug, the compound can be administered prior toadministration of the drug to avoid or ameliorate an allergic responseto the drug. Alternatively, prophylactic administration can be appliedto avoid the onset of symptoms in a patient diagnosed with theunderlying disorder. For example, a compound can be administered to anallergy sufferer prior to expected exposure to the allergen. Compoundsmay also be administered prophylactically to healthy individuals who arerepeatedly exposed to agents known to one of the above-describedmaladies to prevent the onset of the disorder. For example, a compoundcan be administered to a healthy individual who is repeatedly exposed toan allergen known to induce allergies, such as latex, in an effort toprevent the individual from developing an allergy. Alternatively, acompound can be administered to a patient suffering from asthma prior topartaking in activities which trigger asthma attacks to lessen theseverity of, or avoid altogether, an asthmatic episode.

The amount of compound administered will depend upon a variety offactors, including, for example, the particular indication beingtreated, the mode of administration, whether the desired benefit isprophylactic or therapeutic, the severity of the indication beingtreated and the age and weight of the patient, the bioavailability ofthe particular active compound, etc. Determination of an effectivedosage is well within the capabilities of those skilled in the art.

Effective dosages can be estimated initially from in vitro assays. Forexample, an initial dosage for use in animals can be formulated toachieve a circulating blood or serum concentration of active compoundthat is at or above an IC₅₀ of the particular compound as measured in asin vitro assay. Calculating dosages to achieve such circulating blood orserum concentrations taking into account the bioavailability of theparticular compound is well within the capabilities of skilled artisans.For guidance, the reader is referred to Fingl & Woodbury, “GeneralPrinciples,” In: Goodman and Gilman's The Pharmaceutical Basis ofTherapeutics, Chapter 1, pp. 1-46, latest edition, Pergamagon Press, andthe references cited therein.

Initial dosages can also be estimated from in vivo data, such as animalmodels. Animal models useful for testing the efficacy of compounds totreat or prevent the various diseases described above are well-known inthe art. Suitable animal models of hypersensitivity or allergicreactions are described in Foster, (1995) Allergy 50(21Suppl):6-9,discussion 34-38 and Tumas et al., (2001), J. Allergy Clin.Immunol.107(6):1025-1033. Suitable animal models of allergic rhinitisare described in Szelenyi et al., (2000), Arzneimittelforschung50(11):1037-42; Kawaguchi et al., (1994), Clin. Exp. Allergy24(3):238-244 and Sugimoto et al., (2000), Immunopharmacology 48(1):1-7.Suitable animal models of allergic conjunctivitis are described inCarreras et al., (1993), Br. J. Ophthalmol. 77(8):509-514; Saiga et al.,(1992), Ophthalmic Res. 24(1):45-50; and Kunert et al., (2001), Invest.Ophthalmol. Vis. Sci. 42(11):2483-2489. Suitable animal models ofsystemic mastocytosis are described in O'Keefe et al., (1987), J. Vet.Intern. Med. 1(2):75-80 and Bean-Knudsen et al., (1989), Vet. Pathol.26(1):90-92. Suitable animal models of hyper IgE syndrome are describedin Claman et al., (1990), Clin. Immunol. Immunopathol. 56(1):46-53.Suitable animal models of B-cell lymphoma are described in Hough et al.,(1998), Proc. Natl. Acad. Sci. USA 95:13853-13858 and Hakim et al.,(1996), J. Immunol. 157(12):5503-5511. Suitable animal models of atopicdisorders such as atopic dermatitis, atopic eczema and atopic asthma aredescribed in Chan et al., (2001), J. Invest. Dermatol. 117(4):977-983and Suto et al., (1999), Int. Arch. Allergy Immunol. 120(Suppl 1):70-75.Suitable animal models of transplant rejection, such as models of HVGRare described in O'Shea et al., (2004), Nature Reviews Drug Discovery3:555-564; Cetkovic-Curlje & Tibbles, (2004), Current PharmaceuticalDesign 10:1767-1784; and Chengelian et al., (2003), Science 302:875-878.Ordinarily skilled artisans can routinely adapt such information todetermine dosages suitable for human administration.

Dosage amounts will typically be in the range of from about 0.0001 or0.001 or 0.01 mg/kg/day to about 100 mg/kg/day, but can be higher orlower, depending upon, among other factors, the activity of thecompound, its bioavailability, the mode of administration and variousfactors discussed above. Dosage amount and interval can be adjustedindividually to provide plasma levels of the compound(s) which aresufficient to maintain therapeutic or prophylactic effect. For example,the compounds can be administered once per week, several times per week(e.g., every other day), once per day or multiple times per day,depending upon, among other things, the mode of administration, thespecific indication being treated and the judgment of the prescribingphysician. In cases of local administration or selective uptake, such aslocal topical administration, the effective local concentration ofactive compound(s) may not be related to plasma concentration. Skilledartisans will be able to optimize effective local dosages without undueexperimentation.

Preferably, the compound(s) will provide therapeutic or prophylacticbenefit without causing substantial toxicity. Toxicity of thecompound(s) can be determined using standard pharmaceutical procedures.The dose ratio between toxic and therapeutic (or prophylactic) effect isthe therapeutic index. Compounds(s) that exhibit high therapeuticindices are preferred.

Also provided are kits for administration of the thiosemicarbazone orpharmaceutical formulations comprising the compound, that may include adosage amount of at least one thiosemicarbazone or a compositioncomprising at least one thiosemicarbazone as disclosed herein. Kits mayfurther comprise suitable packaging and/or instructions for use of thecompound. Kits may also comprise a means for the delivery of the atleast one thiosemicarbazone or compositions comprising at leastthiosemicarbazone, such as an inhaler, spray dispenser (e.g. nasalspray), syringe for injection or pressure pack for capsules, tables,suppositories, or other device as described herein.

Additionally, the compounds of the present invention can be assembled inthe form of kits. The kit provides the compound and reagents to preparea composition for administration. The composition can be in a dry orlyophilized form, or in a solution, particularly a sterile solution.When the composition is in a dry form, the reagent may comprise apharmaceutically acceptable diluent for preparing a liquid formulation.The kit may contain a device for administration or for dispensing thecompositions, including, but not limited to syringe, pipette,transdermal patch, or inhalant.

The kits may include other therapeutic compounds for use in conjunctionwith the compounds described herein. In one implementation, thetherapeutic agents are immunosuppressant or anti-allergan compounds.These compounds can be provided in a separate form, or mixed with thecompounds of the present invention.

The kits will include appropriate instructions for preparation andadministration of the composition, side effects of the compositions, andany other relevant information. The instructions can be in any suitableformat, including, but not limited to, printed matter, videotape,computer readable disk, or optical disc.

In one implementation, this invention provides a kit comprising acompound selected from the compounds of this invention, packaging, andinstructions for use.

Kits may also be provided that contain sufficient dosages ofthiosemicarbazone or composition to provide effective treatment for anindividual for an extended period, such as a week, 2 weeks, 3, weeks, 4weeks, 6 weeks or 8 weeks or more.

It will be appreciated by one of skill in the art that theimplementations summarized above may be used together in any suitablecombination to generate additional implementations not expressly recitedabove, and that such implementations are considered to be part of thepresent invention.

V. EXAMPLES

The invention is further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims. The following abbreviations were used as noted (Table 3):

TABLE 3 Table of Abbreviations BRIJ ® 35 Polyethylene glycol dodecylether Polyoxyethylene (23) lauryl ether C₁₂H₂₅(OCH₂CH₂)_(n)OH, n~23 DMSOdimethylsulfoxide DTT dithiothreitol EDTA ethylenediaminetetraaceticacid h hour(s) HPLC High-Performance Liquid Chromatography HRMS (ESI)High Resolution Mass Spectrometry (Electrospray Ionization) IC₅₀ halfmaximal inhibitory concentration LC-MS liquid chromatography-massspectrometry MeOH methanol TsOH p-toluenesulfonic acid

A. 1,4-dibenzoylbenzene (1)

Aluminum trichloride (1.37 g, 10.33 mmol) was added to a solution ofterephthaloyl dichloride (1.0 g, 4.42 mmol) in anhydrous dichloromethaneand excess benzene. After stirring for 12 h at room temperature under aninert atmosphere of nitrogen gas, the reaction mixture treated with 10%HCl (50 mL) and the products were extracted with ethyl acetate (3×20mL). The combined organic phases were washed with brine and dried oversodium sulfate. After the organic layer was concentrated under reducedpressure, purification using flash chromatography (silica gel,hexanes:ethyl acetate, 80:20) afforded 1,4-dibenzoyl benzene 1 (0.405 g,1.41 mmol) in a 32% yield.

B. 3-benzoyl benzophenone thiosemicarbazone (3),2-[(3-benzoylphenyl)(phenyl)methylene]-N-methylhydrazinecarbothioamide(27), and2-[(3-benzoylphenyl)(phenyl)methylene]-1-methylhydrazinecarbothioamide(28)

p-toluenesulfonic acid (0.026 g, 0.02 mmol) was added to a solution of1,3 dibenzoylbenzene 2 (2.00 g, 6.98 mmol) in anhydrous methanol (20mL). After stirring at reflux for 10 min, thiosemicarbazide (0.476 g,5.235 mmol) was added to the reaction mixture and stirred for 26 h underan inert atmosphere of nitrogen gas. After 26 h, methanol was removedunder reduced pressure and 50 mL of water was then added. The productswere extracted with ethyl acetate (2×50 mL) and the combined organicphases were washed with brine, dried over anhydrous sodium sulfate, andthe solvent was removed under reduced pressure. Purification using flashchromatography (silica gel, hexanes:ethyl acetate, gradient 90:10 to52:48) afforded 3-benzoyl benzophenone thiosemicarbazone 3 (0.829 g,5.235 mmol) as a light yellow solid in a 44% yield. HRMS (ESI)calculated for C₂₁H₁₇N₃OSH⁺ (M+H)⁺ 360.11651, found 360.11654. Theisolated product 3 was 99.3% pure by HPLC at 300 and 320 nm; retentiontime 7.225 (method: 0-25 min, 50 to 100% acetonitrile, 50 to 0% water).This purified product was submitted for biological testing.

Compounds 27 and 28 are a byproduct obtained in the synthesis ofCompound 3. HRMS (ESI) calculated for C22H20ON3SH+ (M+H) 374.1322, found374.1324. The isolated yield of compounds 27 and 28 were 2.5% and 2.4%,respectively. The location of the attachment of the methyl group has notbeen confirmed.

C. 4-Benzoyl benzophenone thiosemicarbazone (4)

A catalytic amount of p-toluenesulfonic acid was added to a solution of1,4 dibenzoylbenzene 1 (0.286 g, 1 mmol) in anhydrous methanol (10 mL).After stirring at reflux for 10 min, thiosemicarbazide (0.182 g, 2 mmol)was added to the reaction mixture and stirred for 12 h under an inertatmosphere of nitrogen gas. After 12 h, methanol was removed underreduced pressure and 10 mL of water was then added. The products wereextracted with ethyl acetate (2×20 mL) and the combined organic phaseswere washed with brine, dried over anhydrous sodium sulfate, and thesolvent was removed under reduced pressure. Purification using flashchromatography (silica gel, hexanes:ethyl acetate, gradient 70:30)afforded 4-benzoyl benzophenone thiosemicarbazone 4 (0.012 g, 0.033mmol) in a 3.3% yield. HRMS (ESI) calculated for C₂₁H₁₇N₃OSH⁺ (M+H)⁺360.11651, found 360.11649.

D. 3-Benzoyl benzhydrol thiosemicarbazone (6)

1,3 dibenzoylbenzene 2 (2.00 g, 6.98 mmol) was dissolved in anhydrousethanol (20 mL) under an inert atmosphere of nitrogen gas. The reactionmixture was cooled to 0° C. followed by the addition of sodiumborohydride (0.090 g, 2.094 mmol). The reaction mixture was stirred for4 h and quenched the addition of a small amount of water. The reactionmixture was concentrated under reduced pressure and the products wereextracted from water with ethyl acetate (2×50 mL). The combined organicphases were washed with brine, dried over anhydrous sodium sulfate, andthe solvent was removed under reduced pressure. Purification using flashchromatography (silica gel, hexanes:ethyl acetate, gradient 93:7 to30:70) afforded 3-benzoyl benzhydrol 5 (0.601 g, 2.084 mmol) in a 30%yield.

p-toluenesulfonic acid (0.007 g, 0.03 mmol) was added to a solution of3-benzoyl benzyhdrol 5 (0.396 g, 1.09 mmol) in anhydrous tetrahydrofuran(10 mL). After stirring at reflux for 10 min, thiosemicarbazide (0.199g, 2.19 mmol) was added to the reaction mixture and stirred for 2 daysunder an inert atmosphere of nitrogen gas. After 2 days, tetrahydrofuranwas removed under reduced pressure and water (50 mL) was added. Theproducts were extracted with ethyl acetate (2×20 mL) and the combinedorganic phases were washed with brine, dried over anhydrous sodiumsulfate, and the solvent was removed under reduced pressure.Purification using flash chromatography (silica gel, hexanes:ethylacetate, gradient 88:12 to 0:100) afforded 3-benzoyl benzhydrolthiosemicarbazone 6 (0.228 g, 0.631 mmol) as a white solid in a 57%yield. HRMS (ESI) calculated for C₂₁H₁₉N₃OSH⁺ (M+H)⁺ 362.1321, found362.1336.

E. 1-(3-methylbenzoyl),3-(3-methylbenzoyl) benzene thisosemicarbazone(9)

Triethylamine (5.61 mL, 40 mmol) was added dropwise to a solution ofN,O-dimethylhydroxylamine hydrochloride (2.926 g, 30 mmol) in anhydrousdichloromethane (35 mL) at 0° C. After stirring for 10 min, isophthaloyldichloride (2.03 g, 10 mmol) dissolved in 6 mL of anhydrousdichloromethane was added dropwise. The reaction mixture was returned toroom temperature and stirred for 5 h. The reaction mixture was quenchedwith water (50 mL) and the products were extracted with dichloromethane(2×50 mL). The combined organic phases were washed with brine, driedover anhydrous sodium sulfate, and the solvent was removed under reducedpressure. Purification using flash chromatography (silica gel,dichloromethane:methanol, 95:5) affordedN¹,N³-bismethoxy-1,3-benzenedicarboxamide 7 (2.465 g, 9.77 mmol) in a97% yield.

3-bromotoluene (6.684 g, 39.08 mmol) was dissolved in anhydroustetrahydrofuran (30 mL) under an inert atmosphere of nitrogen. Thesolution was stirred for 5 min and cooled to −78° C. followed by thedropwise addition of n-butyllithium in hexanes (2.5M; 13.6 mL). Afterstirring for 3.5 h, the Weinreb amide of isophthaloyl dichloride wasadded dropwise and the solution was stirred for 1 h at −78° C. Thereaction mixture was quenched with 1 M HCl (40 mL) and the products wereextracted with dichloromethane (2×50 mL). The combined organic phaseswere washed with brine, dried over anhydrous sodium sulfate, and thesolvent was removed under reduced pressure. Purification using flashchromatography (silica gel, hexanes:ethyl acetate, gradient, 95:5 to70:30) afforded 2.465 g, 9.77 mmol)1-(3-methylbenzoyl),3-(3-methylbenzoyl) benzene 8 in a 72% yield.

p-toluenesulfonic acid (0.018 g, 0.09 mmol) was added to a solution of1-(3-methylbenzoyl),3-(3-methylbenzoyl) benzene 8 (0.300 g, 0.95 mmol)in anhydrous methanol (10 mL). After stirring at reflux for 10 min,thiosemicarbazide (0.086 g, 0.954 mmol) was added to the reactionmixture and stirred for 12 h under an inert atmosphere of nitrogen gas.After 12 h, methanol was removed under reduced pressure and 10 mL ofwater was then added. The products were extracted with dichloromethane(2×30 mL) and the combined organic phases were washed with brine, driedover anhydrous sodium sulfate, and the solvent was removed under reducedpressure. Purification using flash chromatography (silica gel,hexanes:ethyl acetate, gradient 90:10 to 80:20) afforded1-(3-methylbenzoyl),3-(3-methylbenzoyl) benzene thiosemicarbazone 9(0.063 g, 0.016 mmol) as a white solid in a 17% yield. HRMS (ESI)calculated for C₂₃H₂₁N₃OSH⁺ (M+H)⁺ 388.14781, found 388.14793. Theproduct was determined to be approximately 80% pure and was submittedfor biological testing without further purification.

F. 1,3-bis(2-fluoro-benzoyl)-5-bromobenzene thiosemicarbazone (11)

Triethylamine (5.32 mL, 37.84 mmol) was added dropwise to a solution ofN,O-dimethylhydroxylamine hydrochloride (2.768 g, 28.38 mmol) inanhydrous dichloromethane (45 mL) at 0° C. After stirring for 10 min,2-fluorobenzoyl chloride (0.303 mL, 2.52 mmol) in anhydrousdichloromethane (15 mL) was added dropwise. The reaction mixture wasreturned to room temperature and stirred for 5 h. The reaction mixturewas quenched with water (60 mL) and the products were extracted withdichloromethane (2×60 mL). The combined organic phases were washed withbrine, dried over anhydrous sodium sulfate, and the solvent was removedunder reduced pressure. Purification using flash chromatography (silicagel, hexanes:ethyl acetate, gradient 93:7 to 60:40) afforded2-Fluoro-N-methoxy-N-methyl-benzamide (3.116, 17 mmol) in a 90% yield.

Tert-butyllithium in pentane (1.6M, 7.72 mL) was added dropwise to asolution of 1,3,5 tribromobenzene (0.972 g, 3.09 mmol) in anhydrousether (30 mL) at −78° C. under a flow of nitrogen gas. After 2 h,2-Fluoro-N-methoxy-N-methyl-benzamide (1.132 g, 6.18 mmol) dissolved inanhydrous ether (5 mL) was added dropwise and the reaction mixture wasallowed to slowly come to room temperature and stirred for 24 h. After24 h, the reaction mixture was quenched with water (30 mL) and theproducts were extracted with ether (2×50 mL). The combined organicphases were washed with brine, dried over anhydrous sodium sulfate, andthe solvent was removed under reduced pressure. Purification using flashchromatography (silica gel, hexanes:ethyl acetate, gradient 95:5 to60:40) afforded 2-1,3-bis(2-fluoro-benzoyl)-5-bromobenzene 10 (0.617 g,6.18 mmol) in a 50% yield.

p-toluenesulfonic acid (0.006 g, 0.03 mmol) was added to a solution of1,3-bis(3-fluoro-benzoyl)-5-bromobenzene 10 (0.190 g, 0.473 mmol) inanhydrous tetrahydrofuran (15 mL). After stirring at reflux for 10 min,thiosemicarbazide (0.088 g, 0.97 mmol) was added to the reaction mixtureand stirred for 28 h under an inert atmosphere of nitrogen gas. After 28h, tetrahydrofuran was removed under reduced pressure and 10 mL of waterwas then added. The products were extracted with EtOAc (2×50 mL) and thecombined organic phases were washed with brine, dried over anhydroussodium sulfate, and the solvent was removed under reduced pressure.Purification using flash chromatography (silica gel, hexanes:ethylacetate, gradient 90:11 to 30:70) afforded1,3-bis(2-fluoro-benzoyl)-5-bromobenzene thiosemicarbazone 11 (0.068 g,0.143 mmol) in a 30% yield. HRMS (ESI) calculated for C₂₁H₁₄BrF₂N₃OSH⁺(M+H)⁺ 474.0082, found 474.0087.

G. 1,3-bis-(4-fluorobenzoyl) benzene thiosemicarbazone (13)

Aluminum trichloride (6.53 g, 49.5 mmol) was added to a solution ofisophthaloyl dichloride (5.0 g, 24.8 mmol) in dichloromethane (100 mL).After heating at reflux for 30 min, the monofluorobenzene (2.835 g, 29.5mmol) was added and stirring was continued at reflux. After stirringover 12 h, the reaction mixture was poured onto the crushed ice. Theresulting solution was neutralized with 10% NaOH (100 mL), and extractedwith dichloromethane (3×100 mL). The combined organic layer was washedwith water, followed by brine and dried over Na₂SO₄. After the organiclayer was concentrated under reduced pressure, purification using flashchromatography (silica gel, hexanes:ethyl acetate, 60:40) afforded1,3-bis-(4-fluorobenzoyl) benzene 12 (3.37 g, 10.5 mmol) as a whitesolid in a 42% yield; 1H NMR (500 MHz, CDCl3): δ 8.130 (t, J=1.5 Hz, 1H,ArH), 8.00 (dd, J=7.5 Hz, 1.5 Hz, 1H, ArH), 7.87 (m, 4H, ArH), 7.64 (t,J=8.0 Hz, 1H, ArH), 7.18 (m, 4H, ArH).

1,3-bis-(4-fluorobenzoyl) benzene 12 (0.347 g, 1.08 mmol) was dissolvedin anhydrous methanol (50 mL). The solution was heated at reflux for 15min, and thiosemicarbazide (0.049 g, 0.54 mmol) and a catalytic amountof p-toluenesulfonic acid were added. After 10 h at reflux, theresulting solution was concentrated under reduced pressure. Purificationusing flash chromatography (silica gel, hexanes:ethyl acetate, 70:30)afforded the desired 1,3-bis-(4-fluorobenzoyl) benzene thiosemicarbazone(0.11 g, 0.278 mmol, 52% yield) as a white solid; ¹H NMR (500 MHz,CDCl₃): δ 8.67(s, 1H, NH), 7.97 (m, 1H, ArH), 7.90 (m, 1H, ArH), 7.83(m, 1H, ArH), 7.75 (m, 1H, ArH), 7.68 (m, 1H, ArH), 7.500 (m, 2H, ArH),7.39 (s, 1H, NH ₂), 7.31 (m, 2H, ArH), 7.22 (ddd, J=8.5 Hz, 5.0 Hz, 3.0Hz, 1H, ArH), 7.16 (ddd, J=8.5 Hz, 5.0 Hz, 3.0 Hz, 1H, ArH), 7.06 (ddd,J=8.5 Hz, 5.0 Hz, 3.0 Hz, 1H, ArH), 6.36 (s, 1H, NH ₂); HRMS (ESI)calculated for C₂₁H₁₅F₂N₃OSH (M+H)⁺ 396.0970, found 394.0834; HPLCretention time 13.740, 13.966 min.

H. 1,3-bis-(4-methoxybenzoyl) benzene dithiosemicarbazone (33)

Aluminum trichloride (2.79 g, 21 mmol) was added to a solution ofisophthaloyl dichloride (2.0 g, 10 mmol) in dichloromethane (50 mL).After heating at reflux for 30 min, the anisole (1.29 mL, 11.8 mmol) wasadded and stirring was continued at reflux. After stirring over 20 h,the reaction mixture was poured onto the crushed ice. The resultingsolution was neutralized with 10% NaOH (100 mL), and extracted withdichloromethane (3×100 mL). The combined organic layer was washed withwater, followed by brine, and dried over Na₂SO₄. After the organic layerwas concentrated under reduced pressure, the purification using flashchromatography (silica gel, hexanes:ethyl acetate, 60:40) afforded1,3-bis-(4-methoxy benzoyl) benzene 14 (1.5 g, 4.33 mmol) as a whitesolid in a 43% yield; ¹H NMR (500 MHz, CDCl₃): δ 8.09 (t, J=1.0 Hz, 1H,ArH), 7.95 (dd, J=7.5 Hz, 1.5 Hz, 2H, ArH), 7.84 (ddd, J=8.9, 4.7, 2.5Hz, 4H, ArH), 7.61 (td, J=7.5 Hz, 0.4 Hz, 1H, ArH), 6.97 (ddd, J=8.9,4.9, 2.85 Hz, 4H, ArH), 3.88 (s, 6H, OCH ₃); ¹³C NMR (125 MHz, CDCl₃): δ194.70, 163.53, 138.80, 132.73, 132.60, 1320.61, 129.63, 128.32, 113.76,55.53.

1,3-bis-(4-methoxybenzoyl) benzene 14 (0.45 g, 1.3 mmol) was dissolvedin anhydrous methanol (48 mL). The solution was heated at reflux for 15min, and thiosemicarbazide (0.059 g, 0.64 mmol) and a catalytic amountofp-toluenesulfonic acid were added. After 10 h at reflux, the mixturewas concentrated under reduced pressure. The resulting solid waspurified using flash chromatography (hexanes:ethyl acetate, 60:40) toafford the pure 1,3-bis-(4-methoxybenzoyl) benzene dithiosemicarbazone33 (0.10 g, 0.238 mmol, 37% yield) as a white solid. HRMS (ESI)calculated for C₂₄H₂₅N₆O₂S₂ (M+H)⁺ 493.1475, found 493.1470; HPLCretention time 11.424, 11.664, 11.832 min.

I. 1-(4-hydroxybenzoyl)-3-(4-methoxybenzoyl) benzene thiosemicarbazone(17); 1-(4-methoxybenzoyl)-3-(4-hydroxybenzoyl) benzenethiosemicarbazone (18) and 1-(4-hydroxybenzoyl)-3-(4-methoxybenzoyl)benzene di-thiosemicarbazone (19)

To a well-stirred solution of 1,3-bis-(4-methoxybenzoyl) benzene 14 (700mg, 2.02 mmol) in dichloromethane (25 mL) was added boron trifluoridedimethyl sulfide complex (BF₃.SMe₂, 10 mL). The reaction was stirred for16 h at room temperature, and then quenched with water, and extractedwith ethyl acetate (3×30 mL). The combined organic layer was washed withbrine, dried over Na₂SO₄ and concentrated under reduced pressure. Theresulting crude product was purified using flash chromatography (silicagel, hexanes:ethyl acetate, 50:50) to afford the pure1-(4-hydroxybenzoyl)-3-(4-methoxybenzoyl) benzene 16 (0.2 g, 0.60 mmol)as a white solid in a 30% yield; ¹H NMR (500 MHz, Acetone-d6): δ 9.19(s, OH), 7.89 (dt, J=2 Hz, 0.5 Hz, 1H, ArH), 7.85 (dd, J=7.5 Hz, 1.5 Hz,2H, ArH), 7.72 (ddd, J=10.0 Hz, 5.0 Hz, 3.0 Hz, 2H, ArH), 7.66 (ddd,J=9.5 Hz, 5.0 Hz, 3.0 Hz, 2H, ArH), 7.59 (td, J=7.5 Hz, 0.5 Hz, 1H,ArH), 6.96 (ddd, J=9.5 Hz, 4.5 Hz, 2.5 Hz, 2H, ArH), 6.86 (ddd, J=9.5Hz, 5.0 Hz, 3.0 Hz, 2H, ArH), 3.78 (s, 3H, OCH ₃); ¹³C NMR (125 MHz,Acetone-d₆): δ6193.70, 193.56, 163.61, 161.941, 138.58, 138.35, 132.62,132.41, 132.30, 130.16, 129.67, 128.75, 128.56, 115.25, 113.78, 55.12.

1-(4-hydroxybenzoyl), 3-(4-methoxybenzoyl) benzene 16 (0.20 g, 0.602mmol) was dissolved in anhydrous methanol (10 mL). The solution washeated at reflux for 15 min, and thiosemicarbazide (0.066 g, 0.725 mmol)and a catalytic amount of p-toluenesulfonic acid were added. After 12 hat reflux, the solvent was removed under reduced pressure. Purificationusing flash chromatography (silica gel, hexanes:ethyl acetate, 60:40)afforded the desired a mixture of mono-thiosemicarbazone compounds 17and 18 (0.01 g, 0.0247 mmol, 4% yield) and di-thiosemicarbazone compound19 (0.015 g, 0.0314 mmol, 5.2% yield), both as a light yellow solid.

Compounds 17 and 18: HRMS (ESI) calculated for C₂₂H₁₉N₃O₃SH⁺ (M+H)406.1220, found 406.1221; compound 19: HRMS (ESI) calculated forC₂₃H₂₂N₆O₂S₂H⁺ (M+H) 479.1318, found 479.1319. Compounds 17, 18 and 19:HPLC retention time 8.247, 8.454, 8.586, 8.752 min. The mixture ofcompounds 17, 18 and 19 was collected as two fractions from the samereaction. Fraction 2 is believed to be more pure than fraction 1, basedupon preliminary LC-MS. Further, it is likely that compounds 17, 18 and19 are present in different abundances in fractions 1 and 2. Bothfractions were submitted for biological testing.

J. 1,3-bis-(4-isopropoxybenzoyl) benzene thiosemicarbazone (22)

To a well-stirred solution of 1,3-bis-(4-methoxybenzoyl) benzene 14(1.80 g, 5.2 mmol) in dichloromethane (85 mL) was added borontrifluoride dimethyl sulfide complex (BF₃.SMe₂, 15 mL). The mixture wasstirred for 27 h. After the reaction was quenched by water, the mixturewas extracted with ethyl acetate (3×80 mL). The combined organic layerwas washed with brine, dried over Na₂SO₄, and concentrated under vacuum.The resulting solid was further purified using flash chromatography(silica gel, hexanes: ethyl acetate, 50:50) to afford the pure1,3-bis-(4-hydroxybenzoyl) benzene 20 (0.62 g, 1.95 mmol) as a whitesolid in a 38% yield; ¹H NMR (500 MHz, Acetone-d₆): δ 9.28 (s, OH), 8.04(td, J=1.7 Hz, 1.3 Hz, 1H, ArH), 7.98 (dd, J=7.7 Hz, 1.75 Hz, 2H, ArH),7.80 (ddd, J=9.5 Hz, 4.8 Hz, 2.75 Hz, 4H, ArH), 7.73 (td, J=7.9 Hz, 0.45Hz, 1H, ArH), 7.00 (ddd, J=9.5 Hz, 4.8 Hz, 2.75 Hz, 4H, ArH).

Reactions were conducted using a commercially available microwavereactor (Biotage). In a microwave vial, 1,3-bis-(4-hydroxybenzoyl)benzene 20 (0.310 g, 0.975 mmol), isopropyl bromide (0.851 g, 6.92 mmol)and potassium carbonate (0.955 g, 6.91 mmol) were added to DMF (10 mL),with a magnetic stir bar. The vial was capped tightly and the reactionmixture was heated from r.t. to 90° C. for 2 h. After the reactionmixture was cooled to room temperature, the vial was opened and themixture was transferred to a round bottom flask. The mixture wasquenched with water (50 mL) and extracted with ether (2×50 mL). Theorganic layer was washed with brine, dried over Na₂SO₄, and concentratedunder reduced pressure. The crude product was purified using flashchromatography (silica gel, hexanes: ethyl acetate, 50:50) to afford thepure 1,3-bis-(4-isopropoxybenzoyl) benzene 21 (0.23 g, 0.57 mmol) as awhite solid in a 59% yield; ¹H NMR (500 MHz, Acetone-d₆): δ 8.07 (t,J=1.95 Hz, 1H, ArH), 7.99 (dd, J=7.6 Hz, 1.8 Hz, 2H, ArH), 7.84 (ddd,J=9.6 Hz, 5.2 Hz, 3.0 Hz, 4H, ArH), 7.71 (td, J=8.0 Hz, 0.25 Hz, 1H,ArH), 7.05 (ddd, J=10.1 Hz, 5.3 Hz, 3.2 Hz, 4H, ArH), 4.76 (Septet,J=6.05 Hz, 2H, CH(CH₃)₂), 1.35 (d, J=6.05 Hz, 12H, CH(CH₃) ₂); ¹³C NMR(125 MHz, Acetone-d6): δ 205.23, 193.51, 162.03, 138.41, 132.40, 130.25,129.22, 128.57, 115.07, 69.96, 21.33.

1,3-bis-(4-isopropoxybenzoyl) benzene 21 (0.14 g, 0.39 mmol) wasdissolved in anhydrous methanol (20 mL). The solution was heated atreflux for 15 min, and thiosemicarbazide (0.043 g, 0.47 mmol) and acatalytic amount ofp-toluenesulfonic acid were added. After 10 h atreflux, the solvent was removed under vacuum and the resulting solid wasfurther purified using flash chromatography (silica gel, hexanes: ethylacetate, 50:50) to afford the desired 1,3-bis-(4-isopropoxybenzoyl)benzene thiosemicarbazone 22 as a white solid (0.05 g, 0. 105 mmol, 27%yield); HRMS (ESI) calculated for C₂₇H₂₉N₃O₃SH⁺(M+H) 476.2002, found476.2005; HPLC retention time 18.440, 18.767 min.

K. 1,3-bis-(4-isopropoxybenzoyl) benzene dithiosemicarbazone (23)

1,3-bis-(4-isopropoxybenzoyl) benzene 21 (0.092 g, 0.255 mmol) wasdissolved in anhydrous methanol (12 mL). The solution was heated atreflux for 15 min, and thiosemicarbazide (0.019 g, 0.21 mmol) and acatalytic amount of p-toluenesulfonic acid were added. After 8 h atreflux, the solvent was removed under vacuum and the resulting solid wasfurther purified using flash chromatography to (silica gel, hexanes:ethyl acetate, 50:50) afford the desired 1,3-his-(4-isopropoxybenzoyl)benzene dithiosemicarbazone 23 as a light yellow solid (0.02 g, 0. 042mmol, 16% yield); HRMS (ESI) calculated for C₂₈H₃₂N₆O₂S₂H⁺ (M+H)549.2101, found 549.2103; HPLC retention time 10,788, 11.355, 11.525min.

L. 1,3-bis-(4-bromobenzoyl) benzene thiosemicarbazone (24)

To a flask containing aluminum trichloride (1.36 g, 10.2 mmol) undernitrogen was added bromobenzene (15 mL, 142.8 mmol). Isophthalylchloride(1.00 g, 4.88 mmol) was dissolved in a minimal amount ofbromobenzaldehyde and added to the reaction flask via syringe. Thereaction stirred at reflux for 6 h, after which it was stirred at roomtemperature for 12 h, followed by another 3 h at reflux. The reactionwas quenched with H₂O (20 mL). The resulting mixture was then added to50 mL of 10% HCl cooled in an ice bath. The mixture was extracted withethyl acetate (3×10 mL) and the combined organic layers were washedsequentially with deionized water, dilute HCl, deionized water, andbrine. Crude product crashed out of the organic layers as a white solidand was collected via filtration. Recrystallization of the solid fromethyl acetate afforded 1,3-bis-(4-bromobenzoyl) benzene 23. in 31%yield. ¹H NMR (DMSO-d₆, 500 MHz) δ 8.14-8.13 (m, 1H, ArH), 7.95 (dd,J=7.5 Hz, 1.5 Hz, 2H, ArH), 7.70-7.64 (m, 9H, ArH).

1,3-bis-(4-bromobenzoyl) benzene 23 (0.550 g, 1.21 mmol) was dissolvedin dry THF (20 mL). The solution was heated at reflux for 15 min, andthiosemicarbazide (0.220 g, 2.42 mmol) and a catalytic amount ofp-toluenesulfonic acid (0.023 g, 0.121 mmol) were added. After 24 h atreflux, the result solution was concentrated under reduced pressure andthe residue was dissolved in dichloromethane (25 mL). The organic layerwas washed with deionized water (15 mL), dried over Na₂SO₄, andconcentrated. The crude product was purified via column chromatographyto give the double condensation product 24 in <1% yield (6.7 mg, 11.3μmol). Only the double condensation product was able to be isolated. ¹HNMR (DMSO-d₆, 500 MHz) δ 9.87 (2H, bs, NH), 8.46 (2H, brs, NH), 8.29(2H, brs, NH), 7.84 (1H, t, J=7.5 Hz, ArH), 7.64 (4H, d, J=8.5 Hz, ArH),7.56 (4H, d, J=7.5 Hz, ArH), 7.48 (2H, dd, J=7.5 Hz, 1.5 Hz, ArH), 7.20(1H, s, ArH). HRMS (ESI) calculated for C₂₂H₁₈Br₂N₆S₂H⁺ (M+H)⁺ 588.9474,found 588.9461.

M. 1,3,5-Tribenzoyl benzene thiosemicarbazone (25)

To a round bottom flask containing methanol (16 mL) was added1,3,5-tribenzoylbenzene (0.250 g, 0.640 mmol). This was stirred undernitrogen and heated to reflux. Thiosemicarbazide (0.05536 g, 0.608 mmol)was then added to the flask, followed by p-toluenesulfonic acid (0.00122g, 0.0064 mmol). The reaction mixture was stirred at reflux for 14 h.The crude mixture was purified using column chromatography, eluting with35% ethyl acetate in hexanes, however the product still containedimpurities and was submitted for testing without further purification.HRMS (ESI) calculated for C₂₈H₂₁O₂N₃SH⁺ (M+H)⁺ 464.14272, found464.14267. Compound 25 was estimated to be approximately 80% pure byNMR, and was submitted for biological testing without furtherpurification.

N. Biological Activity

Human liver Cathepsin L (Sigma) was preincubated with test compounds atvarious concentrations for 5 minutes at 25° C. The assay was initiatedby addition of substrate Z-Phe-Arg-aminomethylcoumarin (“Z-F-R-AMC”Bacchem) and the final assay conditions were 1 nM cathepsin L, 50 μMZ-F-R-AMC, 100 mM sodium acetate pH 5.5, 1 mM EDTA (Omnipure), 3 mM DTT(EMD), 0.01% BRIJ 35 (Sigma), and 2.0% DMSO (Acros). Test compounds wereserially diluted with DMSO and water to include a final concentrationrange of 10 μM to 10 pM. The reaction was monitored fluorometrically for5 minutes at 25° C. using black 96-well Corning 3686 assay microplateswith a Thermo Fluoroskan Ascent FL microplate reader at excitation andemission filter wavelengths of 355 nm and 460 nm, respectively. Datawere analyzed to determine IC₅₀ values utilizing GraphPad Prism 4.03software with a minimum of a triplicate on the same microplate.

Recombinant human procathepsin K was obtained from Enzo Life Sciences.Activation of the proenzyme was performed in 32.5 mM sodium acetate pH3.5, EDTA 1 mM, NaCl 500 mM, human procathepsin K 5.5 □M, at roomtemperature. Activation times were optimized and varied between 35 and150 minutes. Cathepsin K was preincubated with test compounds at variousconcentrations for 5 minutes at 25° C. The assay was initiated byaddition of substrate Z-Phe-Arg-aminomethylcoumarin (“Z-F-R-AMC”Bacchem) and the final assay conditions were 1.5 nM cathepsin K, 50 μMZ-F-R-AMC, 150 mM sodium acetate pH 5.5, 2.5 mM EDTA (Omnipure), 2.5 mMDTT (EMD), 0.01% BRIJ 35 (Sigma), and 4.0% DMSO (Acros). Test compoundswere serially diluted with DMSO and water to include a finalconcentration range of 10 μM to 10 pM. The reaction was monitoredfluorometrically for 5 minutes at 25° C. using black 96-well Corning3686 assay microplates with a Thermo Fluoroskan Ascent FL microplatereader at excitation and emission filter wavelengths of 355 nm and 460nm, respectively. Data were analyzed to determine IC₅₀ values utilizingGraphPad Prism 4.03 software with a minimum of a triplicate on the samemicroplate.

Human liver Cathepsin B (Calbiochem) was pre-incubated with testcompound at various concentrations for 5 minutes at 37° C. The assay wasinitiated by addition of substrate Z-Arg-Arg-aminomethylcoumarin(“Z-R-R-AMC” Bacchem) and the final assay conditions were 1.1 nMcathepsin B, 60 μM Z-R-R-AMC, 126 mM sodium potassium phosphate pH 6.0(Fisher), 0.3 mM EDTA (Omnipure), 2.7 mM DTT (Omnipure), 0.004% BRIJ 35(Sigma), and 2.0% DMSO (Acros) in a final volume of 200 μL. Testcompounds were serially diluted with DMSO and 0.01% BRIJ 35 to include afinal concentration range of 20 μM to 10 pM. The reaction was monitoredfluorometrically for 5 minutes at 37° C. using black 96-well Corning3686 assay microplates with a Thermo Fluoroskan Ascent FL microplatereader at excitation and emission filter wavelengths of 355 nm and 460nm, respectively. Data were analyzed to determine IC50 values utilizingGraphPad Prism 4.03 software with a minimum of a triplicate on the samemicroplate.

TABLE 4 Biological Activity IC₅₀ Compound Cathepsin L (nM) Cathepsin K(nM) Cathepsin B (nM)  3 10.5 17.4 >10,000  6 23.8 nd >10,000  9 7,8231,034 nd 11 8.12 nd >10,000 13 24.3 8,500 >10,000 17, 18 & 19 >10,0006,162 >10,000 (fraction 1) 17, 18 & 19 >10,000 3,204 >10,000 (fraction2) 22 >10,000 nd >10,000 23 >10,000 >10,000 nd24 >10,000 >10,000 >10,000 25 25.0 2,796 nd 33 587 2,105 >10,000

What is claimed is:
 1. A method of inhibiting an activity of acathepsin, comprising contacting the cathepsin with a compound having aformula I, in an amount of effective to inhibit an activity of thecathepsin,

or a solvate or pharmaceutically acceptable salt thereof, wherein X isselected from the group consisting of C(═O), CH(OR⁶) and

n is 0, 1, 2 or 3; m is 0, 1, 2 or 3; p is 0, 1, 2 or 3; R¹ is hydrogen,C₁-C₃ alkyl, aryl and arylalkyl; R² is hydrogen or C₁-C₃ alkyl; each R³and R⁵ independently is selected from the group consisting of hydroxyl,C₁-C₃ alkyl, C₁-C₂ alkoxy, fluoro, and chloro; each R⁴ independently isselected from the group consisting of hydroxyl, C₁-C₆ alkyl, C₁-C₆alkoxy, halo, amino, nitro, nitroso and acyl; and R⁶ is selected fromthe group consisting of hydrogen and methyl.
 2. The method of claim 1,wherein the cathepsin is cathepsin K or cathepsin L.
 3. The method ofclaim 1, further comprising: contacting the cathepsin in vitro with thecompound having formula I.
 4. The method of claim 1, further comprisingcontacting the cathepsin in a cell with the compound having formula I.5. The method of claim 1, wherein X is C(═O) or CH(OR⁶).
 6. The methodof claim 5, wherein X is C(═O).
 7. The method of claim 1, wherein m iszero.
 8. The method of claim 7, wherein n is zero and p is zero.
 9. Themethod of claim 1, wherein n is zero and p is zero.
 10. The method ofclaim 1, wherein m is
 1. 11. The method of claim 10, wherein R⁴ is haloor acyl.
 12. The method of claim 11, wherein R⁴ is substitutedaryl-C(O)— or unsubstituted aryl-C(O)—.
 13. The method of claim 11,wherein R⁴ is benzoyl.
 14. The method of claim 1, wherein each of n andp, independently, is zero or one; and each of R³ and R⁵, independently,is selected from the group consisting of hydroxyl, methyl, methoxy andfluoro.
 15. The method of claim 1, wherein the compound having theformula I has a formula II, or a solvate or pharmaceutically acceptablesalt thereof:


16. The method of claim 5, wherein X is C(═O).
 17. The method of claim16, wherein each of n, m and p, independently, is zero or one.
 18. Themethod of claim 16, wherein n is one and R³ is selected from the groupconsisting of hydroxyl, methyl, methoxy, and fluoro.
 19. The method ofclaim 16, wherein p is one and R⁵ is selected from the group consistingof hydroxyl, methyl, methoxy and fluoro.
 20. The method of claim 16,wherein m is one and R⁴ is selected from the group consisting of haloand acyl.
 21. The method of claim 15, wherein X is

and m is zero or one.
 22. A compound of claim 21, wherein each of n andp, independently, is zero or one.
 23. The method of claim 15, whereinthe compound is 3-benzoyl benzophenone thiosemicarbazone (3) having theformula:


24. The method of claim 15, wherein n=1, m=0, and p=1.
 25. The method ofclaim 24, wherein R³ and R⁵ are fluoro.
 26. The method of claim 25,wherein the compound is 1,3-bis-(4-fluorobenzoyl) benzenethiosemicarbazone (13) having the formula:


27. The method of claim 15, wherein n=0, m=1, and p=0.
 28. The method ofclaim 27, wherein R⁴ is benzoyl.
 29. The method of claim 28, wherein thecompound is 1,3,5-Tribenzoyl benzene thiosemicarbazone (25) having theformula: